Physico-Chemical Characteristics of Date Seed Oil Grown in Sudan

Problem statement: Studies were conducted on the physical-chemical pro perties of Sudanese date seed oil extracted from two date palm cultivar s (Phoenix dactylifera L.) ALBarakawi and Alqundeila. The extracted oil from ALBarakawi seeds weighed 6.833% w/w oil, Alqundeila oil extracted weighed 5.064% w/w. Approach: The physical properties of ALBarakawi oil were: viscosity: 17cp, refractive Index: 1.444, density: 0.9116 g cm −3 color: Within the range 1. 6-11.1 (red-yellow). Fo r Alqundeila the physical properties were: viscosity: 20cp, ref ractive index: 1.458, density: 0.9174 g cm −3 color: 1.8-12.1 (red yellow). Results: The chemical properties were: Acid value 2.55, sapo nfication value 0.255 mg g, iodine value 71.12 gm I 2/100 gm fat, peroxide value: 4.8 mg. Peroxide/Kgoil f r ALBarakawi. For Alqundeila they were: acid value 2.47, saponfication value 0. 267 mg g and iodine value 83.31gm I 2/100 gm fat, peroxide value: 7.4 mg Peroxide/Kgoil. In this study, High Performance Liquid Chromatography (HPLC) was utilized for the i dentification and quantification of vitamin E in these samples. The content of vitamin E for ALBarakawi was 5.821 and 6.054 ppm for Alqundeila. For the fatty acid content in these oils, Gas Chromatog r phy-Mass Spectrometry (GC-MS) was used. The constituents of fatty acids (calculated % relative to total fatty acid constituents) in ALBarakawi were: Saturated lauric acid 37.10%, palmitic acid: 9.24%, Stearic acid 1.71%, Unsaturated fatty acids: Linol eic acid (Omega-6) 4.33%, Oleic acid (Omega-9) 32.66%. For Alqundeila, the found fatty acids were: Saturated lauric acid o.11%, Palmitic acid: o.42%, Stearic acid 46.93%. Conclusion/Recommendations: The elements types and contents in these oils were: Iron 0.27 ppm, Magnesium 0.204 ppm, Sodium 60 ppm, Potassium 470 ppm, Calcium 25 ppm, Selenium 34 .4 ppb for ALBarakawi. The contents of these elements in Alqundeila were: iron 0.27 ppm Magnesium 0.07 ppm, Potassium 2.3 ppm, Selenium 12.6 ppb. The methods involved in this study covered, Ga s Chromatography-Mass Spectrometry (GC-MS), High Performance Liquid Chromatography (HPLC), Atom ic Absorption Spectroscopy (AAS) and Refractometry. Physico-chemical properties of date se d oil reported in this study were found similar to most of the early published results in this field.


INTRODUCTION
Return to the natural sources of various food constituents (vitamins, minerals, fats, proteins….). Is becoming the target for researchers throughout the world.This return is caused by the growing recognition of hazards to the human health by foods of non-natural source. The palm tree date forms one of the sources of food for humans and animals in addition to its economic benefits. Most of the Mediterranean and Arabian Peninsula countries are considered date producing countries. Sudan is also ranked among these countries. Different types of local palm trees are grown in Sudan. They are named ALBarakawi-Alqundeila-ALMishriq wad Khatib-wad Laqai-ALTamudhah AL Kullimah. The fruit of the date is composed of a fleshy peri-carp and seed. The date seeds as in most counties was finding little use as no research work was done on its possible useful constituents. However recent work including work done by (Besbes et al., 2005;2004a;2004b;Chiew et al., 2007;Devshony et al., 1992) proved that date seeds have extractable high value added components. The potential uses of date seed and its constituents are expected to cover uses in cosmetics, medicine, pharmaceuticals, feed and food industries (Harry, 1936;Salem and Hegazi, 1971;E1-Shurafa et al., 1982;Whaibi et al., 1985;Whaibi and Basalah, 1986;Mossa et al., 1986). The literature survey revealed that no previous work was done on Sudanese palm tree seeds oil to evaluate its physico-chemical characteristics or any other possible potential applications. The aim of this work was to extract date seed oil and study its quality characteristics in two selected palm tree types named ALBarakawi and Alqundeila. This work was also intended to stimulate further research on date seed oil to cover stability, phenolic compounds contents and to point out a constituent that can be considered a marker for the different type palm of trees in Sudan.

Materials:
Seeds collection: The seeds of two cultivars: ALBarakawi and Alqundeila were collected from a local date's market at Omdurman city. (Major source of the collected dates is Northern Sudan).

Extraction methods:
Oil extraction and preparation: Weights of about 750-900g of ALBarakawi and Alqundeila seeds were used for oil extraction. The seeds were separated from the fruits, dried and crushed into minute granules using automatic machine (retsch-100) for 3 m. Total lipids were extracted from the seeds with hexane using Soxhlet apparatus. The extraction process continued for 4-6 h. The solvent was evaporated on rotary evaporator under reduced pressure and the produced oil (free from nhexane) was collected, weighed, stored in a dark container in a deep-freeze (-20°C) till subsequent analyses.

Fatty acids composition:
The oil fatty acid methyl esters were prepared as described in Christie (AOAC, 1990). One ml of the produced oil was added to seven ml methanolic NaoH (0.5 M) followed by seven ml of methanolic H 2 SO 4 before shaking. The solution was left overnight. Two ml of n-hexane or heptane was added to the reaction mixture followed by saturated NaCl. This mixture was shaken well and allowed to separate into two layers. One ml from the upper layer was transferred to a new tube and dried with anhydrous Na 2 SO 4. Analytical methods: Viscosity determination: Viscosity was determined at 30°C using HAAKE viscometer 6 plus (thermoelectron corporation instrument type 387-0100).

Determination of Refractive index:
The Refractive index was determined by using BELLINGHAM and STANLEY L TD LONDON-(No 918095 MADE in ENGLAND, instrument). Refractometer was adjusted first, using distilled water.

Colour determination:
The Colour was determined by using Lovibond or Tintometer typed" instrument (Tintometer L TD . The colour laboratory Salisbury England).

Vitamin E identification and determination by HPLC and GC-mass:
Vitamin E in the oil sample (0.2 gm mL −1 solution in n-hexane) was identified and evaluated by HPLC and GC-mass. The quantification of Vitamin E in the oils was determined using standard vitamin E solution containg 2000 ppm diluted to obtain a calibration curve ranging between 20-2000 ppm. (n-hexane + THF + 2-propanol) (500 + 30 + 2) GC-Mass system: Analyses were carried out using a Varian 450 Gas chromatography equipped with Varian 220-IT Mass spectrometer and VF-5 ms capillary column (30×0.25 mm; 0.25 um film thickness). The oven temperature was held at 50°C for 5 min and programmed at 7°C/min to 300°C for 5 min. Other operating conditions were: Helium gas carrier at a flow rate of 1.0 mL −1 min. injector and detector temperature were 250 and 280°C respectively; split ratio 1:50. Mass spectra were taken at 70 ev mass ranges from m/z 40-400 amu.

GC-MS identification and determination of fatty acids methyl esters in the oils: Gas Chromatography
Mass Spectrometer was performed using Shimadzu instrument (GC-MS-QP-2010) fitted with electron impact (EI 1.70 ev) mode. The analytical column was RTX 5 (5% phenyl-95% dimethyl polysiloxane with length of 30 m ×0.25 µm). Helium gas was used as a carrier gas at a flow rate of 1 mL min −1 . the temperature was programmed at 40°C for 0 min then increased to 150°C at the rate of 15°C/min with hold time 5 min then increased to 250°C with hold time 10 min. The temperature of injector was 250°C.
AAS identification and determination of elements in the oils: Atomic Absorption Spectroscopy was performed using Shimadzu instrument (AAS-6800).
For the Mg, Fe, Ca, Na, K, 10 mL oil sample was dissolved in 1 mL carbon tetra Chloride (CCL 4 ). To this solution 10 mL −1 10% v/v nitric acid was added, mixed well for about 10 m before centrifugation at 2500-3000 revolution per minuets for 2 m. The supernatant was then separated and the nominated elements were determined by AAS with reference to their respective standards. For selenium the same above procedure was followed but using nickel sulphate 0.25 gm dissolved in 10 mL of 10% v/v nitric acid (Price, 1979). Table  1 reflects the physico-chemical characteristics of the extracted seeds oil of the two date palm cultivars (ALBarakawi and Alqundeila) compared with characteristics of some reported values cited in the Literature (Besbes et al., 2005;Devshony et al., 1992).

RESULTS
The oil was a semi-solid at temperatures below 10°C and a viscous liquid at a ambient temperature.

DISCUSSION
In this study it was found that the chemical constituents which are of interest were the fatty acids constituents and vitamin E content. The seeds oil can be considered a good source of lauric acid and Oleic acid (Omega-9). This is in agreement with the finding reported by (Besbes et al., 2005) for α-tocopheral in their extracted seeds oils Table 1. It is worth noting that the content of Oleic acid and lauric acid in the Sudanese date seed oils were 37.10 and 32.66% respectively at a ratio of 1.136. On the date seed oils reported by Devshony et al. (1992), they were (42.5%) Oleic acid compared to (21%) lauric acid at a ratio2:1. Table 1 also reflects that ALBarakawi summarised physico-chemical properties out-weigh those of Alguendela with the exception of stearic acid contents the other chemical constituent identified in the date seed oils was the naturally occurring antioxidant, vitamin E (tocopherols). Medically, vitamin E is a potent antioxidant that protects the body against oxidation reactions (radicals) that damage membranes cholesterol transporting lipoproteins. Other medical use cover activity as screening reagent protecting against skin damage and aging by UV radiation, inhibits growth of cancer cells and a protector against at herosclerosis by lowering cholesterol level (Packer, 1991;Traber et al., 1997;Sundram et al., 1989;Goh et al., 1994;Lercker and Rodriguez-Estrada, 2000;Ziegler and Filer, 1996;Rimm et al., 1993). The richest source of vitamins E or tocopherols in the diet is vegetable oils and their products including palm oil (Chiew et al., 2007). Tocopherol in date tocopherols in date seed oil are reported by (Besbes et al., 2005). In our study the date seed oil was also found to contain Vit E ( Fig. 2a and b). The semi-solid nature of the oils is an indication of the presence of major saturated and unsaturated fatty acids (lauric acid and oleica cid respectively) The presence of α-tocopherols in the oils is reflected by the coinciding retention times of 2.5 min. for the standard and a corresponding peak in the sample chromatogram ( Fig. 2a and 2b). sample chromatogram also shows a number of unidentified peaks which could include (α, β, γ, δ) tocopherols.The content of αtocopherols was estimated in this study to be about 6.054 ppm in both studied seed oils compared to a 25-40 ppm reported by (Besbes et al., 2005) for α-tocopheral in their extracted seeds oils Table 1. Table 3 shows the mass spectral assigments of major Vit E fragments depicted from Fig. 3.
The molecular ion at 430 m/z corresponds to the molecular weight of tocopherol (430). The other fragments presented in the table collectively indicate the tocopherol structure.